Synthesis and use of ω-hydroxylated polyunsaturated fatty acids

ABSTRACT

The present invention provides a synthetic chemical method for preparing ω-hydroxylated polyunsaturated fatty acids (PUFAs) including 20-hydroxyeicosatetraenoic acid (20-HETE), 20-hydroxyeicosapentaenoic acid (20-HEPE), and 22-hydroxydocosahexaenoic acid (22-HDoHE) and a method of use thereof for treating cancer and macular degeneration.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/275,293, filed on Feb. 13, 2019, which is the national phase under 35U.S.C. § 371 of International Application No. PCT/US2017/047113, filedon Aug. 16, 2017, which claims priority benefit to U.S. ProvisionalApplication No. 62/375,526, filed on Aug. 16, 2016, the entirety ofwhich are incorporated herein by reference for all purpose.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under NIEHS grant R01ES02710, NIEHS Superfund Basic Research Program grant P42 ES04699, NIHLBgrant HL059699 and NINDS grant U54 NS079202. The government has certainrights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Polyunsaturated fatty acids (PUFAs) are metabolized by CYP450 enzymesthat function as monooxygenases mainly by catalyzing hydroxylation andepoxidation (see Ref 1). Arachidonic acid (ARA) is metabolized by theCYP450s to hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoicacids (EETs). While all EET isomers are almost equally formed,20-hydroxyeicosatetraenoic acid (20-HETE), a product by w-hydroxylation,is major HETE regioisomer derived from ARA. These metabolites areimportant lipid mediators that play important roles in various diseases(see Ref. 2). Interestingly, EETs and HETEs often have opposingbiological functions (see Ref. 2).

Every CYP450 produces both EETs and HETEs. However, epoxygenases, suchas CYP2C and CYP2J isoforms, produce mostly epoxides, and hydroxylases,such as CYP4A and CYP4F isoforms, produce mostly 20-HETE. The same CYPisoforms also metabolize ω-3-polyunsaturated fatty acids such aseicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to thecorresponding epoxy- and hydroxyl polyunsaturated fatty acids (see Ref3). Recently, beneficial effects from dietary supplements of fish oilincluding prescription ω-3 fish oil have triggered interests in thebiological functions of their metabolites (see Ref 4). For example, itis showed that epoxides of EPA and DHA can reduce pain perception (seeRef. 5), blood pressure (see Ref. 6), and angiogenesis (see Ref. 7).Herein, the present disclosures are concentrating on the biological roleof ω-hydroxy polyunsaturated fatty acid metabolites.

Biological roles of 20-HETE have been well studied, and have been shownto have detrimental effects on several diseases such as hypertension(see Ref 8), cancer (see Ref. 9), and cardiovascular and kidney diseases(see Ref 10). In addition, there is growing evidence to suggest that20-HETE is a potent agonist of the transient receptor potentialvanilloid receptor 1 (TRPV1) which is activated by endogenous lipidmediators and is closely related to pain (see Ref. 11).

However, little is known about the biological roles of otherω-hydroxylated PUFAs due mostly to their limited availability ordifficulty to synthesize them. These are 20-hydroxyeicosapentaenoic acid(20-HEPE) and 22-hydroxydocosahexaenoic acid (22-HDoHE) derived from EPAand DHA respectively. 22-hydroxydocosahexaenoic acid (22-HDoHE) is anendogenous lipid mediator produced by cytochrome P450 ω-hydroxylases(largely CYP4A and CYP4F) from docosahexaenoic acid (DHA, 22:6 ω-3), asshown in FIG. 1A. It is an endogenous compound and has been detected inmany tissues such as brain, lung, kidney and liver, as shown in FIG. 1B(see Ref 35).

Gopal et. al. reported a chemical synthesis of 20-HETE by aSuzuki-Miyaura cross-coupling of a cis-vinylbromide compound with afunctionalized borane (see In Tetrahedron Lett. 2004, 45 (12),2563-2565). Apart from that, several biosynthesis of 20-HETE have beenreported (see Ref 12). Harmon et. al. reported a 20-HEPE synthesis byoxygenation of ω-3 fatty acids by human cytochrome P450 4F3B (seeProstaglandins, leukotrienes, and essential fatty acids 2006, 75 (3),169-77). However, a complete synthesis of 22-HDoHE has not been reportedyet.

In view of the number of possible applications for the treatment ofdiseases there is a need for a synthetic chemical method of preparing22-HDoHE, 20-HEPE, and 20-HETE.

Described herein, inter alia, are solutions to these and other problemsin the art.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method forpreparing a compound of Formula II:

the method including:

-   -   forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III:

and

-   -   a compound of Formula IV:

-   -   under conditions suitable to form the compound of Formula II;        wherein:    -   the transition-metal coupling agent comprises a metal selected        from the group consisting of copper, iron, nickel, palladium,        zinc, and combinations thereof;    -   R is C₁-C₄ alkyl;    -   X is selected from the group consisting of Cl, Br, I, —OTs, and        —OTf;    -   subscript m is 2 or 3;    -   subscript n is 0 or 1; and    -   bond a is a single bond or a triple bond.

In one aspect, the present invention is directed to a method forpreparing a compound of Formula I:

the method including:

-   -   i) forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III:

and

-   -   a compound of Formula IV:

-   -   under conditions suitable to form a compound of Formula II:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II, a deactivated palladium catalyst, a        deactivating agent, and hydrogen, under conditions suitable for        hydrogenation to form the compound of Formula I;        wherein:    -   the transition-metal coupling agent comprises a metal selected        from the group consisting of copper, iron, nickel, palladium,        zinc, and combinations thereof;    -   R is C₁-C₄ alkyl;    -   X is selected from the group consisting of Cl, Br, I, —OTs, and        —OTf;    -   subscript m is 2 or 3;    -   subscript n is 0 or 1; and    -   bond a and bond b are each a single bond or bond a is a triple        bond and bond b is a double bond.

In another aspect, the present invention is directed to a method ofpreparing a ω-hydroxylated polyunsaturated fatty acid having a structureof:

or a salt thereof;the method including:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula

and

-   -   a compound of Formula Iv-1 or Iv-2:

-   -   under conditions suitable to form a corresponding compound of        Formula II-1a or II-2a:

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-1a or II-2a, Lindlar catalyst, a        N-containing 5- to 10-membered heteroaryl compound, a C₂-C₆        alkene, and hydrogen, under conditions suitable for        hydrogenation to form a corresponding compound of Formula I-1a        or I-2a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-1a or I-2a and an alkali hydroxide under        conditions suitable for saponification to form the corresponding        the compound or the salt of 20-HETE or 20-HEPE;        wherein:    -   the copper ion is a copper (I) ion salt selected from the group        consisting of CuI, CuBr, CuCl, and Cu(OAc); and    -   X is selected from the group consisting of Cl, Br, I, and —OTs.

In another aspect, the present invention is directed to a method ofpreparing a ω-hydroxylated polyunsaturated fatty acid having a structureof:

or a salt thereof;the method includes:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-2a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form the compound of Formula II-3a:

-   -   ii) forming a reaction mixture comprising a compound of Formula        II-3a, Lindlar catalyst, a N-containing 5- to 10-membered        heteroaryl compound, a C₂-C₆ alkene, and hydrogen, under        conditions suitable for hydrogenation to form a compound of        Formula I-3a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-3a and an alkali hydroxide under        conditions suitable for saponification to form the compound or        the salt of 22-HdoHE;        wherein:    -   the copper ion is a copper (I) ion salt selected from the group        consisting of CuI, CuBr, CuCl, and Cu(OAc); and    -   X is selected from the group consisting of Cl, Br, I, and —OTs.

In still another aspect, the present invention is directed to apharmaceutical composition including a ω-hydroxylated polyunsaturatedfatty acid, or an ester form thereof, and a pharmaceutically acceptableexcipient.

In yet another aspect, the present invention is directed to a method oftreating cancer or macular degeneration, the method includingadministering to a subject in need thereof an effective amount of aω-hydroxylated polyunsaturated fatty acid, or the ester form thereof, ora pharmaceutical composition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the conversion of docosahexaenoic acid (DHA) to22-hydroxydocosahexaenoic acid (22-HDoHE) through the metabolismcatalyzed by cytochrome P450 ω-hydroxylases. FIG. 1B is a bar graphshowing the tissue levels of 22-HDoHE in mouse organs.

FIG. 2A shows LC-MS/MS analysis of 20-hydroxyeicosatetraenoic acid(20-HETE) from a commercial source vs. synthesized. FIG. 2B showsLC-MS/MS analysis of 20-hydroxyeicosapentaenoic acid (20-HEPE). FIG. 2Cshows LC-MS/MS analysis of 22-HDoHE. Extracted-ion chromatogram (XIC):319.2/275.1 for 20-HETE, 317.2/255.0 for 20-HEPE, and 343.2/281.1 for22-HDoHE, respectively.

FIG. 3 is ¹H NMR spectra of 20-HETE, 20-HEPE, and 22-HDoHE.

FIG. 4A is a set of graphs of calcium influx in heterologously expressedmTRPV1 as a function of time induced by capsaicin, 20-HEPE, 22-HDoHE and20-HETE. FIG. 4B is a bar graph of the quantification of the Ca²⁺responses (area under curve, AUC) induced by capsaicin, 20-HEPE,22-HDoHE and 20-HETE.

FIG. 5 is a bar graph of the COX inhibitory activity of 20-HETE, 20-HEPEand 22-HDoHE. Less than 0% inhibition was obtained with eicosapentaenoicacid (EPA), suggesting that EPA is a substrate for COX-2.

FIG. 6A is a graph showing results of von Frey mechanical nociceptiveassays. FIG. 6B is a graph showing a dose-response manner only withipsilateral administration of 20-HETE.

FIG. 7A is a simplified scheme of the animal experiment: Matrigelcontaining VEGF (angiogenesis inducer) and 22-HDoHE or vehicle wereinjected into C57BL/6 mice, after 4-7 days of treatment, the Matrigelplugs were dissected and subjected to angiogenesis analysis. FIG. 7B isrepresentative images of dissected Matrigel plugs: in the VEGF control(positive control), Matrigels had strong infiltration of blood into theplugs, which were abolished by the 22-HDoHE treatment. FIG. 7C is aquantification of angiogenesis in the dissected Matrigel plugs, whichshowed that 22-HDoHE strongly inhibited VEGF-induced angiogenesis invivo.

FIG. 8 is a series of photographs showing 22-HDoHE inhibition ofVEGF-induced endothelial cell migration in HUVEC cells. The cellmigration assay was conducted using a standard Boyden chamber assay with10 ng/mL VEGF as chemoattractant, and the assay time was 6 hours.

FIG. 9 is a photograph and a bar graph showing systematic treatment of0.5 mg/kg/day 22-HDoHE inhibited ˜50% of B16F10 melanoma growth inC57BL/6 male mice. B16F10 melanoma cells (200,000 cells per mouse) weresub-Q injected into C57BL/6 mice to induce primary tumor growth ofmelanoma, then the mice were treated with 22-HDoHE using mini-pumps at adose of 0.5 mg/kg/day. After 2-wk treatment, the mice were sacrificed todissect the tumors for analysis.

FIG. 10 is a series of photographs showing treatment with 22-HDoHEinhibits tube formation in HMVEC-dLy cells.

FIG. 11 is a series of photographs showing 22-HDoHE inhibits cellularmigration of lymphatic endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides a method of preparing ω-hydroxylatedpolyunsaturated fatty acids (i.e., 20-HETE, 20-HEPE, and 22-HDoHE)through a convergent synthesis approach. Serial copper-mediated couplingreactions to construct skipped alkynes and their partial hydrogenationto the desired cis-double bonds of each ω-hydroxylated polyunsaturatedfatty acid have been used as key reactions. The convergent synthesisminimizes time and effort since several intermediates are partiallyshared during the preparation of all three ω-hydroxylatedpolyunsaturated fatty acids. Finally, hydrolysis of the esters of20-HETE, 20-HEPE, and 22-HDoHE has provided the desired ω-hydroxylatedpolyunsaturated fatty acids in good overall yields, respectively.

The present invention provides a method of using ω-hydroxylatedpolyunsaturated fatty acids for treatment of cancer or maculardegeneration. The ω-hydroxylated polyunsaturated fatty acids, forexample 22-HDoHE, have demonstrated to inhibits tumor growth,angiogenesis, lymphangiogenesis in cell culture and animal models.

II. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. (i.e., C₁-C₄ meansone to four carbons). For example, C₁₋₄ alkyl includes, but is notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, and tert-butyl.

The term “alkene” is a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond and having the numberof carbon atoms indicated (i.e., C₂-C₆ means two to six carbons).Examples of alkenes include, but are not limited to, ethene, propene,isopropene, 1-butene, 2-butene, isobutene, butadiene, 1-pentene,2-pentene, isopentene, 2-methyl-2-butene, 1,3-pentadiene,1,4-pentadiene, 1-hexene, 2-hexene, 3-hexene, 1,3-hexadiene,1,4-hexadiene, 1,5-hexadiene, 2,4-hexadiene, or 1,3,5-hexatriene.

The term “alkyne” is a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one triple bond.

“Amine” refers to a compound having formula N(R)₃ where the R groups canbe hydrogen, alkyl, aryl, or heteroalkyl, among others. The R groups canbe the same or different. For example, the amines can be primary amine(two R is each hydrogen), secondary amine (one R is hydrogen), andtertiary amine (each R is other than hydrogen). In some embodiments, thesecondary amine is a cyclic amine where two R groups bond to thenitrogen atom form a 5-6 membered heterocyclic ring. Non-limitingexamples of cyclic amines include pyrrolidine, piperidine, andmorpholine. In other embodiments, the amine is a diamine where one of Rgroups is an aminoalkyl. Non-limiting examples of diamines includeethylenediamine.

“Alkyl amine” refers to an amine as defined above where the R groups areone or more alkyl groups. For example, the alkylamine can bemonoalkylamine, dialkylamine or trialkylamine. Monoalkylamines useful inthe present invention include, but are not limited to, ethylamine,propylamine, isopropylamine, butylamine, ethylenediamine, andethanolamine. Dialkylamines useful in the present invention include, butare not limited to, diethylamine, dipropylamine, diisopropylamine, anddibutylamine. Trialkylamines useful in the present invention include,but are not limited to, trimethylamine, triethylamine, tripropylamine,and triisopropylamine.

The term “N-containing heteroaryl” refers to a monocyclic or fusedbicyclic aromatic ring assembly containing 5 to 10 ring atoms, where atleast one atom of the ring is N. The term “N-containing 5- to10-membered heteroaryl” refers heteroaryl groups having from 5 to 10ring members and from 1 to 3 ring atoms including N, O or S. The“N-containing 5- to 10-membered heteroaryl” compounds include, but arenot limited to, pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, indole,isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, andphthalazine. In some embodiments, the “N-containing 5- to 10-memberedheteroaryl” include heteroaryl groups having from 5 to 10 ring membersand only nitrogen heteroatoms, such as pyrrole, pyridine, imidazole,pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-,1,2,4- and 1,3,5-isomers), indole, isoindole, quinoline, isoquinoline,quinoxaline, quinazoline, and phthalazine. In other embodiments, the“N-containing 5- to 10-membered heteroaryl” include heteroaryl groupshaving from 5 to 10 ring members and only one nitrogen heteroatom, suchas pyrrole, pyridine, indole, isoindole, quinoline, and isoquinoline.

The abbreviation “—OTs” refers to p-toluenesulfonate. The abbreviation“—OTf” refers to trifluoromethanesulfonate.

The term “metal” refers to elements of the periodic table that aremetallic and that can be neutral, or positively charged as a result ofhaving more or fewer electrons in the valence shell than is present forthe neutral metallic element. Metals useful in the present inventioninclude the alkali metals and transition metals. Alkali metals in thepresent invention include alkali metal cations. Alkali metal cationsuseful in the present invention include Li⁺, Na⁺, K⁺, and Cs⁺.Transition metals useful in the present invention include Sc, Ti, V, Cr,Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, La, Hf,Ta, W, Re, Os, Ir, Pt, Au, Hg and Ac. Transition metals useful in thepresent invention include transition metal cations, for example, Cd²⁺,Co²⁺, Co³⁺, Cr²⁺, Cr⁺, Cu⁺ (i.e., Cu(I)), Cu²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Mn³⁺,Ni²⁺, Ni³⁺, Pd²⁺ (i.e., Pd(II)), and

The term “base” refers to a functional group that deprotonates water toproduce a hydroxide ion. Exemplary bases are amines as defined above,N-containing heteroaryl as defined above, or alkali carbonates. Examplesof alkali carbonates include potassium carbonate, sodium carbonate, andcesium carbonate.

The term “alkali carbonate” refers to a class of chemical compoundswhich are composed of an alkali metal cation and the carbonate anion(—CO₃ ²⁻). Alkali carbonates useful in the present invention includesodium carbonate (Li₂CO₃), sodium carbonate (Na₂CO₃), potassiumcarbonate (K₂CO₃), and cesium carbonate (Cs₂CO₃).

The term “alkali hydroxide” refers a class of chemical compounds whichare composed of an alkali metal cation and the hydroxide anion (OH⁻).Alkali hydroxides useful in the present invention include LiOH, NaOH,KOH, and CsOH.

The term “catalyst” refers to a substance that increases the rate of achemical reaction by reducing the activation energy, but which is leftunchanged by the reaction. Catalysts may be classified as eitherhomogeneous or heterogeneous. A homogeneous catalyst is one whosemolecules are dispersed in the same phase as the reactant molecules. Aheterogeneous catalyst is one whose molecules are not in the same phaseas the reactants, which are typically gases or liquids that are adsorbedonto the surface of the solid catalyst. Catalysts useful in the presentinvention are both homogeneous catalysts and heterogeneous catalysts.

The term “transition-metal coupling agent” refers to a compound that iscomposed of a transition metal as defined above that can be neutral, orpositively charged. The transition-metal coupling agent plays a criticalrole in a transition metal mediated cross-coupling reaction to form acarbon-carbon bond.

The term “deactivated palladium catalyst” refers a palladium (0)catalyst that is poisoned with addition of various forms of lead andsulfur, certain metal oxides, N-containing heteroaryl compounds.Examples of “catalyst poisons” include, but are not limited to, theaddition of lead acetate, lead oxide, quinolone, sulfides, thiols, orcombinations thereof to the catalyst. Example of a commercialdeactivated palladium catalyst is a Lindlar catalyst. The Lindlarcatalyst is used for the hydrogenation of alkynes to alkenes (i.e.without further reduction into alkanes).

The term “deactivating agent” refers compounds that are used to furtherpoison the deactivated palladium catalyst. Examples of “deactivatingagent” include N-containing 5- to 10-membered heteroaryl compounds,thiols, or diamines. Non-limiting examples of N-containing 5- to10-membered heteroaryl compounds include pyridine, picoline, lutidine,collidine, and quinoline. Non-limiting examples of thiols include3,6-dithia-1,8-octanediol. Non-limiting examples of diamines includeethylenediamine.

The term “aprotic solvent” refers to solvents that lack an acidichydrogen. Consequently, they are not hydrogen bond donors. Commoncharacteristics of aprotic solvents are solvents that can accepthydrogen bonds, solvents do not have acidic hydrogen, and solventsdissolve salts. Example of aprotic solvents include, but are not limitedto, N-methylpyrrolidone (NMP), tetrahydrofuran (THF), ethyl acetate(EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethylsulfoxide (DMSO), propylene carbonate (PC), and hexamethylphosphoramide(HMPA).

The term “forming a reaction mixture” refers to the process of bringinginto contact at least two distinct species such that they mix togetherand can react, either modifying one of the initial reactants or forminga third, distinct, species, a product. It should be appreciated,however, the resulting reaction product can be produced directly from areaction between the added reagents or from an intermediate from one ormore of the added reagents which can be produced in the reactionmixture.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product, which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and deleterious to the recipient thereof.

The term “pharmaceutically acceptable excipient” and “pharmaceuticallyacceptable carrier” refer to a substance that aids the administration ofan active agent to and absorption by a subject and can be included inthe compositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g. decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. In some embodiments, inhibition meansnegatively affecting (e.g. decreasing) the concentration or levels ofthe protein relative to the concentration or level of the protein in theabsence of the inhibitor. In some embodiments, inhibition refers toreduction of a disease or symptoms of disease. In some embodiments,inhibition refers to a reduction in the activity of a particular proteintarget. Thus, inhibition includes, at least in part, partially ortotally blocking stimulation, decreasing, preventing, or delayingactivation, or inactivating, desensitizing, or down-regulating signaltransduction or enzymatic activity or the amount of a protein. In someembodiments, inhibition refers to a reduction of activity of a targetprotein resulting from a direct interaction (e.g. an inhibitor binds tothe target protein). In other embodiments, inhibition refers to areduction of activity of a target protein from an indirect interaction(e.g. an inhibitor binds to a protein that activates the target protein,thereby preventing target protein activation).

The terms “treat”, “treating”, or “treatment” refers to any indicia ofsuccess in the therapy or amelioration of an injury, disease, pathologyor condition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. The term“treating” and conjugations thereof, may include prevention of aninjury, pathology, condition, or disease. In some embodiments, treatingis preventing. In other embodiments, treating does not includepreventing.

The term “patient” or “subject in need thereof” refers to a livingorganism suffering from or prone to a disease or condition that can betreated by administration of a pharmaceutical composition as providedherein. Non-limiting examples include humans, other mammals, bovines,rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals. In some embodiments, a patient is human.

A “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). Anexample of an “effective amount” is an amount sufficient to contributeto the treatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). A“prophylactically effective amount” of a drug is an amount of a drugthat, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of an injury, disease, pathology or condition, or reducingthe likelihood of the onset (or reoccurrence) of an injury, disease,pathology, or condition, or their symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations. An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount,” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an enzyme or protein relative to the absence of theantagonist. The exact amounts will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached. Dosage amounts and intervals can be adjusted individually toprovide levels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal)compatible with the preparation. Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals (e.g. humans), includingleukemia, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound or method provided herein include brain cancer, glioma,glioblastoma, neuroblastoma, prostate cancer, colorectal cancer,pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, lungcancer, and cancer of the head. Exemplary cancers that may be treatedwith a compound or method provided herein include cancer of the thyroid,endocrine system, brain, breast, cervix, colon, head & neck, liver,kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus, Medulloblastoma, colorectal cancer, pancreaticcancer. Additional examples include, Hodgkin's Disease, Non-Hodgkin'sLymphoma, multiple myeloma, neuroblastoma, glioma, glioblastomamultiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, primary brain tumors, cancer, malignantpancreatic insulanoma, malignant carcinoid, urinary bladder cancer,premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms ofthe endocrine or exocrine pancreas, medullary thyroid cancer, medullarythyroid carcinoma, melanoma, colorectal cancer, papillary thyroidcancer, hepatocellular carcinoma, or prostate cancer.

When introducing elements of the present disclosure, the articles “a”,“an”, “the”, and “said” are intend to mean that there are one or more ofthe elements. The terms “comprising”, “including”, and “having” areintended to be inclusive and mean that there may be additional elementsother than the listed elements.

III. Method of Preparing Compounds

A. Alkynes of Formula II

In one aspect, provided herein is a method for preparing a compound ofFormula II:

the method including:

-   -   forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III:

and

-   -   a compound of Formula IV:

-   -   under conditions suitable to form the compound of Formula II;        wherein:    -   the transition-metal coupling agent comprises a metal selected        from the group consisting of copper, iron, nickel, palladium,        zinc, and combinations thereof;    -   R is C₁-C₄ alkyl;    -   X is selected from the group consisting of Cl, Br, I, —OTs, and        —OTf;    -   subscript m is 2 or 3;    -   subscript n is 0 or 1; and    -   bond a is a single bond or a triple bond.

Transition-metal coupling agent can be a compound that includes one ormore transition metals or transition metal cations. Non-limitingexamples of transition metals include Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu,Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir,Pt, Au, Hg and Ac. Non limiting examples of transition metal cationsinclude Cd²⁺, Co²⁺, Co³⁺, Cr²⁺, Cr³⁺, Cu⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Mn³⁺,Ni²⁺, Ni³⁺, Pd²⁺, and Zn²⁺. In some embodiments, the transition-metalcoupling agent includes a metal selected from the group consisting ofcopper (Cu), iron (Fe), nickel (Ni), palladium (Pd), zinc (Zn), andcombinations thereof.

In some embodiments, the transition-metal coupling agent includescopper. In some embodiments, the transition-metal coupling agent iscopper. In some embodiments, the transition-metal coupling agentincludes a copper ion. In some embodiments, the transition-metalcoupling agent is a copper ion.

In some embodiments, the copper ion is a copper (I) ion.

In some embodiments, the copper (I) ion is a copper (I) salt.Non-limiting examples of the copper (I) salt include CuI, CuBr, CuCl,and Cu(OAc). In some embodiments, the copper (I) ion is a copper (I)salt selected from the group consisting of CuI, CuBr, CuCl, and Cu(OAc).In some embodiments, the copper (I) salt is CuI. In some embodiments,the copper (I) salt is CuBr. In other embodiments, the copper (I) saltis CuCl. In still other embodiments, the copper (I) salt is Cu(OAc)(i.e., copper (I) acetate).

The carbon-carbon cross coupling of the terminal alkyne of Formula (IV)with the compound of Formula (III) can also be conducted in a reactionmixture that includes a palladium catalyst, a Cu(I) ion as a ω-couplingagent, and a base. In those embodiments, the transition-metal couplingagent includes a palladium catalyst and a copper (I) ion. The palladiumcatalyst, for example, is a zerovalent palladium (i.e., Pd(0)) complex.Examples of such palladium catalysts include a palladium-phosphinecomplex in which palladium is ligated to phosphines (e.g., Pd(PPh₃)₄).Palladium (II) is often employed as a pre-catalyst since it exhibitsgreater stability than Pd(0) over an extended period of time and can bestored under normal laboratory conditions for months. The Pd(II)catalyst is reduced to Pd(0) in the reaction mixture by either an amine,a phosphine ligand, or a reactant, allowing the reaction to proceed. Insome embodiments, the palladium-phosphine complex is a palladium(II)-phosphine complex. A commonly used palladium (II)-phosphate complexis Pd(PPh₃)₂Cl₂. In those specific embodiments, the transition-metalcoupling agent includes Pd(PPh₃)₂Cl₂ and a copper (I) ion. Otherpalladium-phosphine complexes include bidentate ligand (e.g.,1,2-Bis(diphenylphosphino)ethane (dppe),1,3-Bis(diphenylphosphino)propane (dppp), and1,1′-Bis(diphenylphosphino)ferrocene (dppf)) palladium complexes, forexample Pd(dppe)Cl and Pd(dppp)Cl₂, and Pd(dppf)Cl₂.

The transition metal coupling agent including a copper (I) ion can bepresent in the coupling reaction mixture in an amount of from 0.5 to 2molar equivalents, 1 to 1.5 molar equivalents, 1 to 1.3 molarequivalents, 1 to 1.2 molar equivalents, or 1.0 molar equivalent,relative to the compound of Formula (IV). In some embodiments, thecopper (I) ion is present in the coupling reaction mixture in an amountof from 0.5 to 2 molar equivalents, 1 to 1.5 molar equivalents, 1 to 1.3molar equivalents, 1 to 1.2 molar equivalents, or 1.0 molar equivalent,relative to the compound of Formula (IV). In some embodiments, CuI ispresent in the coupling reaction mixture in an amount of from 0.5 to 2molar equivalents, 1 to 1.5 molar equivalents, 1 to 1.3 molarequivalents, 1 to 1.2 molar equivalents, or 1.0 molar equivalent,relative to the compound of Formula (IV). In some other embodiments, CuIis present in the coupling reaction mixture in an amount of 1.0 molarequivalent, relative to the compound of Formula (IV).

In general, the coupling reaction can include a base in the reactionmixture. Without being bond by theory, it is believed that the base inthe reaction mixture is to neutralize the hydrogen halide,p-toluenesulfonic acid, or trifluoromethanesulfonic acid that isproduced as the byproduct of this coupling reaction. In someembodiments, the base is an amine or an alkali carbonate. The amines canbe alkylamine compounds, for example, di-(C₁-C₄ alkyl)amines and atri-(C₁-C₄ alkyl)amines. The amine can also be cyclic amines, forexample, pyrrolidine, piperidine, and morpholine. In some embodiments,the base is selected from the group consisting of a di-(C₁-C₄alkyl)amine, a tri-(C₁-C₄ alkyl)amine, and an alkali carbonate.Non-limiting examples of di-(C₁-C₄ alkyl)amines include diethylamine,dipropylamine, diisopropylamine, and dibutylamine. Non-limiting examplesof tri-(C₁-C₄ alkyl)amines include trimethylamine, triethylamine,tripropylamine, and triisopropylamine. In some embodiments, the base istriethylamine. In other embodiments, the base is diethylamine. In someembodiments, the base is an alkali carbonate. The alkali carbonate canbe potassium carbonate, sodium carbonate, or cesium carbonate. In someembodiments, the alkali carbonate is potassium carbonate or cesiumcarbonate. In other embodiments, the alkali carbonate is cesiumcarbonate. In some selected embodiments, the base is cesium carbonate.

The base can be present in excess in the coupling reaction mixture. Insome embodiments where the base is an amine, the amine is present inexcess in the coupling reaction mixture. In some embodiments, the basecan be present in the coupling reaction mixture in an amount of from 0.5to 10 molar equivalents, 0.5 to 5 molar equivalents, 1 to 2 molarequivalents, 1 to 1.5 molar equivalents, or 1.0 molar equivalent,relative to the compound of Formula (IV). In some embodiments, thealkali carbonate is present in the coupling reaction mixture in anamount of from 0.5 to 2 molar equivalents, 1 to 1.5 molar equivalents, 1to 1.3 molar equivalents, 1 to 1.2 molar equivalents, or 1.0 molarequivalent, relative to the compound of Formula (IV). In someembodiments, cesium carbonate is present in the coupling reactionmixture in an amount of from 0.5 to 2 molar equivalents, 1 to 1.5 molarequivalents, 1 to 1.3 molar equivalents, 1 to 1.2 molar equivalents, or1.0 molar equivalent, relative to the compound of Formula (IV). In someother embodiments, cesium carbonate is present in the coupling reactionmixture in an amount of 1.0 molar equivalent, relative to the compoundof Formula (IV).

When di-(C₁-C₄ alkyl)amines or tri-(C₁-C₄ alkyl)amines are used as thebase, they can be sometimes used as solvents. For example, triethylamineor diethylamine can be used as the solvent as well as the base in thereaction mixture for the coupling reaction.

The coupling reaction mixture further includes an iodide salt. When X isI and the copper (I) salt is CuI, an iodide salt can be optional in thereaction mixture. The iodide salt can be sodium iodide (NaI) orpotassium iodide (KI). In some embodiments, the coupling reactionmixture further includes an iodide salt selected from the groupconsisting of sodium iodide and potassium iodide. In some embodiments,the coupling reaction mixture further includes potassium iodide. Inother embodiments, the coupling reaction mixture further includes sodiumiodide.

The iodide salt can be present in the coupling reaction mixture in anamount of from 0.5 to 2 molar equivalents, 1 to 1.5 molar equivalents, 1to 1.3 molar equivalents, 1 to 1.2 molar equivalents, or 1.0 molarequivalent, relative to the compound of Formula (IV). In someembodiments, potassium iodide is present in the coupling reactionmixture in an amount of from 0.5 to 2 molar equivalents, 1 to 1.5 molarequivalents, 1 to 1.3 molar equivalents, 1 to 1.2 molar equivalents, or1.0 molar equivalent, relative to the compound of Formula (IV). In someother embodiments, potassium iodide is present in the coupling reactionmixture in an amount of 1.0 molar equivalent, relative to the compoundof Formula (IV). In some embodiments, sodium iodide is present in thecoupling reaction mixture in an amount of from 0.5 to 2 molarequivalents, 1 to 1.5 molar equivalents, 1 to 1.3 molar equivalents, 1to 1.2 molar equivalents, or 1.0 molar equivalent, relative to thecompound of Formula (IV). In some other embodiments, sodium iodide ispresent in the coupling reaction mixture in an amount of 1.0 molarequivalent, relative to the compound of Formula (IV).

In some embodiments, the coupling reaction mixture further includes anaprotic solvent. Example of aprotic solvents include, but are notlimited to, N-methylpyrrolidone (NMP), tetrahydrofuran (THF), ethylacetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN),dimethyl sulfoxide (DMSO), and hexamethylphosphoramide (HMPA). In someembodiments, the aprotic solvent is dimethylformamide.

As described above, the base such as di-(C₁-C₄ alkyl)amines ortri-(C₁-C₄ alkyl)amines can be sometimes used as solvents. In thoseembodiments, the coupling reaction mixture does not include anadditional aprotic solvent.

The coupling reaction can be carried out at a molar concentration offrom 0.1 to 2 mol/L, 0.2 to 1.5 mol/L, 0.2 to 1.0 mol/L, 0.2 to 0.5mol/L, or 0.3 to 0.5 mol/L, based on the compound of Formula (IV). Insome embodiments, the molar concentration of the compound of Formula(IV) in the coupling reaction mixture reaction is from 0.1 to 2 mol/L,0.2 to 1.5 mol/L, 0.2 to 1.0 mol/L, 0.2 to 0.5 mol/L, or 0.3 to 0.5mol/L. In some embodiments, the molar concentration of the compound ofFormula (IV) in the coupling reaction mixture reaction is from 0.2 to1.0 mol/L, 0.2 to 0.5 mol/L, or 0.3 to 0.5 mol/L. In other embodiments,the molar concentration of the compound of Formula (IV) in the couplingreaction mixture reaction is from 0.3 to 0.5 mol/L.

In some embodiments, the compound of Formula III is a compound ofFormula III-1:

In some embodiments, the compound of Formula IV is a compound of FormulaIV-1:

In some embodiments, the compound of Formula II is selected from thegroup consisting of:

For example, non-limiting examples of C₁-C₄ alkyl include methyl, ethyl,isopropyl, and t-butyl. In some embodiments, R is methyl. In someembodiments, R is ethyl. In other embodiments, R is t-butyl.

In some embodiments, X is Cl, Br, I, or —OTs. In some embodiments, X isCl. In other embodiments, X is Br. In other embodiments, X is I. Instill other embodiments, X is —OTs.

In various embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-1:

and

-   -   a compound of Formula IV-1:

-   -   under conditions suitable to form a compound of Formula II-1:

wherein the transition-metal coupling agent, the base, R, and X are asdefined and described herein.

In various embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-1:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-2:

wherein the transition-metal coupling agent, the base, R, and X are asdefined and described herein.

In various embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-2:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-3:

wherein the transition-metal coupling agent, the base, R, and X are asdefined and described herein.

In some selected embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a copper ion, an        iodide salt, an alkali carbonate, an aprotic solvent, a compound        of Formula III-1a:

and

-   -   a compound of Formula IV-1:

-   -   under conditions suitable to form a compound of Formula II-1:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, and X are as defined and described herein.

In some selected embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a copper ion, an        iodide salt, an alkali carbonate, an aprotic solvent, a compound        of Formula III-1a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-2a:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, and X are as defined and described herein.

In some selected embodiments, the method including:

-   -   forming a coupling reaction mixture comprising a copper ion, an        iodide salt, an alkali carbonate, an aprotic solvent, a compound        of Formula III-2a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-3a:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, and X are as defined and described herein.

In one selected embodiment, the copper (I) ion salt is CuI.

In one selected embodiment, the iodide salt is sodium iodide.

In one selected embodiment, the alkali carbonate is cesium carbonate.

In one selected embodiment, the aprotic solvent is dimethylformamide.

In one selected embodiment, X is Br.

In general, the coupling reaction is carried out at room temperature.Elevated reaction temperature can be used to accelerate the reaction,especially on a large scale process. Initial cooling the reactionmixture may also be used on a large scale process. For example, thereaction temperature can be from 10° C. to 50° C., 20° C. to 40° C., 20°C. to 30° C., or about 25° C.

B. Alkenes of Formula I

In one aspect, provided herein is a method for preparing a compound ofFormula I:

the method including:

-   -   i) forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III:

and

-   -   a compound of Formula IV:

-   -   under conditions suitable to form a compound of Formula II:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II, a deactivated palladium catalyst, a        deactivating agent, and hydrogen, under conditions suitable for        hydrogenation to form the compound of Formula I;        wherein:    -   the transition-metal coupling agent comprises a metal selected        from the group consisting of copper, iron, nickel, palladium,        zinc, and combinations thereof;    -   R is C₁-C₄ alkyl;    -   X is selected from the group consisting of Cl, Br, I, —OTs, and        —OTf;    -   subscript m is 2 or 3;    -   subscript n is 0 or 1; and    -   bond a and bond b are each a single bond or bond a is a triple        bond and bond b is a double bond.

R is as described in detail above. In one selected embodiments, R ismethyl.

X is as described in detail above. In one selected embodiment, X is Br.

The transition-metal coupling agent is as described in detail above. Inone selected embodiment, the transition metal coupling agent includesCuI.

The base is as described in detail above. In one selected embodiment,the base is cesium carbonate.

In some embodiments, the coupling reaction mixture further includes aniodide salt. The iodide salt is as described in detail above. In oneselected embodiment, the iodide salt is sodium iodide.

In some embodiments, the coupling reaction mixture further includes anaprotic solvent. The aprotic solvent is as described in detail above. Inone selected embodiment, the aprotic solvent is dimethylformamide.

The deactivated palladium catalysts can be prepared by poisoning apalladium catalyst with addition of various forms of lead and sulfur,N-containing heteroaryl compounds. Examples of “catalyst poisons”include, but are not limited to, the addition of lead acetate, leadoxide, quinolone, sulfides, thiols, or combinations thereof to thecatalyst. Example of a commercial deactivated palladium catalyst isLindlar catalyst. In some selected embodiments, the deactivatedpalladium catalyst is Lindlar catalyst.

In general, the deactivating agent is used to further poison thedeactivated palladium catalyst in a reaction mixture. Furtherdeactivating the deactivated palladium catalyst (e.g., Lindlar catalyst)is known to enhance the hydrogenation selectivity of alkynes to alkenes,preventing formation of alkanes. Examples of the deactivating agentsinclude N-containing 5- to 10-membered heteroaryls, thiols, or diamines.Non-limiting examples of N-containing 5- to 10-membered heteroarylsinclude pyridine, picoline, lutidine, collidine, and quinolone.Non-limiting examples of thiols include 3,6-dithia-1,8-octanediol.Non-limiting examples of diamines include ethylenediamine. In someembodiments, the deactivating agent is selected from the groupconsisting of pyridine, quinoline, and ethylenediamine. In one selectedembodiment, the deactivating agent includes pyridine. In anotherselected embodiment, the deactivating agent is pyridine.

In some embodiments of the hydrogenation, the hydrogenation reactionmixture further includes a C₂-C₆ alkene. Without being bond by theory,further addition of alkenes is found to improve the hydrogenationselectivity of alkynes (i.e., the compound of Formula II) to alkenes(i.e., the compound of Formula I), preventing formation of alkanes. Ingeneral, a branched C₅-C₆ non-terminal alkene is preferred. Non-limitingexamples of branched non-terminal C₅-C₆ alkenes include 2-pentene,isopentene, 2-methyl-2-butene, 2-hexene, 2-methyl-2-pentene,2,3-dimethyl-2-butene, (E)-3-methyl-2-pentene, (Z)-3-methyl-2-pentene,(E)-4-methyl-2-pentene, (Z)-4-methyl-2-pentene, and 3-hexene. In oneselected embodiment, the C₁-C₆ alkene is 2-methyl-2-butene. In someselected embodiments of the hydrogenation, the hydrogenation reactionmixture further includes 2-methyl-2-butene.

Alkyne hydrogenation is known to be regioselective, occurring via synaddition to give the cis-alkene. Therefore, the hydrogenation of thecompound of Formula II provides the compound of Formula I in allcis-configurations.

In various embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-1:

and

-   -   a compound of Formula IV-1:

-   -   under conditions suitable to form a compound of Formula II-1:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-1, a deactivated palladium catalyst, a        deactivating agent, and hydrogen, under conditions suitable for        hydrogenation to form the compound of Formula I-1:

wherein the transition-metal coupling agent, the base, the deactivatedpalladium catalyst, the deactivating agent, R, and X are as defined anddescribed herein.

In various embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-1:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-2:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-2, a deactivated palladium catalyst, a        deactivating agent, and hydrogen, under conditions suitable for        hydrogenation to form the compound of Formula I-2:

wherein the transition-metal coupling agent, the base, the deactivatedpalladium catalyst, the deactivating agent, R, and X are as defined anddescribed herein.

In various embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a        transition-metal coupling agent, a base, a compound of Formula        III-2:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-3:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-3, a deactivated palladium catalyst, a        deactivating agent, and hydrogen, under conditions suitable for        hydrogenation to form the compound of Formula II-3:

wherein the transition-metal coupling agent, the base, the deactivatedpalladium catalyst, the deactivating agent, R, and X are as defined anddescribed herein.

In some selected embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-1a:

and

-   -   a compound of Formula IV-1:

-   -   under conditions suitable to form a compound of Formula II-1a:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-1a, Lindlar catalyst, a N-containing 5-        to 10-membered heteroaryl compound, a C₂-C₆ alkene, and        hydrogen, under conditions suitable for hydrogenation to form        the compound of Formula I-1a:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, the N-containing 5- to 10-membered heteroaryl compound,the C₂-C₆ alkene, and X are as defined and described herein.

In some selected embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-1a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-2a:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-2a, Lindlar catalyst, a N-containing 5-        to 10-membered heteroaryl compound, a C₂-C₆ alkene, and        hydrogen, under conditions suitable for hydrogenation to form        the compound of Formula I-2a:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, the N-containing 5- to 10-membered heteroarylscompound, the C₂-C₆ alkene, and X are as defined and described herein.

In some selected embodiments, the method including:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-2a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form a compound of Formula II-3a:

and

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-3a, Lindlar catalyst, a N-containing 5-        to 10-membered heteroaryl compound, a C₂-C₆ alkene, and        hydrogen, under conditions suitable for hydrogenation to form        the compound of Formula I-3a:

wherein the copper ion, the iodide salt, the alkali carbonate, theaprotic solvent, the N-containing 5- to 10-membered heteroaryl compound,the C₂-C₆ alkene, and X are as defined and described herein.

In one selected embodiment, X is Br.

In one selected embodiment, the copper ion includes CuI.

In one selected embodiment, the iodide salt is sodium iodide.

In one selected embodiment, the base is cesium carbonate.

In one selected embodiment, the aprotic solvent is dimethylformamide.

In one selected embodiment, the N-containing 5-10 heteroaryl compound ispyridine.

In one selected embodiment, the C₂-C₆ alkene is 2-methyl-2-butene.

In general, the hydrogenation reaction is carried out at roomtemperature under hydrogen atmosphere. Suitable solvents for thehydrogenation include, but are not limited to, an alcohol (e.g.,methanol or ethanol) and ethyl acetate. The hydrogen pressure for thereaction can be, for example one atmosphere.

C. Omega-Hydroxylated Polyunsaturated Fatty Acids

In some embodiments of preparing the compound of Formula I, the methodfurther includes the preparation of ω-hydroxylated polyunsaturated fattyacid having a structure of:

or a salt thereof,the method including:

-   -   forming a hydrolysis reaction mixture comprising an alkali        hydroxide and the compound of Formula I-1, 1-2, or 1-3:

-   -   under conditions suitable for saponification to form the        corresponding 20-HETE, 20-HEPE, or 22-HdoHE, or a salt thereof;        wherein R is methyl or ethyl.

Suitable alkali hydroxides include LiOH, NaOH, and KOH. In someembodiments, the alkali hydroxide is selected from the group consistingof LiOH, NaOH, and KOH. In one selected embodiment, the alkali hydroxideis LiOH. In another selected embodiment, the alkali hydroxide is NaOH.

Suitable solvents for saponification include, but are not limited to,water, an alcohol (e.g., methanol or ethanol), ether (e.g., THF), or amixture thereof. In some embodiments, when R is methyl, the hydrolysisreaction mixture further includes methanol. In other embodiments, when Ris ethyl, the hydrolysis reaction mixture further includes ethanol. Insome other embodiments, the hydrolysis reaction mixture further includesa mixture of water and THF.

In general, the hydrolysis of the ester is carried out at roomtemperature.

In other embodiments of preparing the compound of Formula I wherein R ist-butyl, the method further includes forming a deprotection reactionmixture comprising the compound of Formula I-1, I-2, or I-3 and a strongacid under conditions suitable for deprotecting the t-butyl group toform the corresponding 20-HETE, 20-HEPE, or 22-HdoHE. Suitable strongacids include, but are not limited to, HCl, sulfuric acid, andtrifloroacetic acid.

In one aspect, provided herein is a method of preparing a ω-hydroxylatedpolyunsaturated fatty acid having a structure of:

or a salt thereof,the method including:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-1a:

and

-   -   a compound of Formula Iv-1 or Iv-2:

-   -   under conditions suitable to form a corresponding compound of        Formula II-1a or II-2a:

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-1a or II-2a, Lindlar catalyst, a        N-containing 5- to 10-membered heteroaryl compound, a C₂-C₆        alkene, and hydrogen, under conditions suitable for        hydrogenation to form a corresponding compound of Formula I-1a        or I-2a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-1a or I-2a and an alkali hydroxide under        conditions suitable for saponification to form the corresponding        20-HETE or 20-HEPE;        wherein:    -   the copper ion is a copper (I) ion salt selected from the group        consisting of CuI, CuBr, CuCl, and Cu(OAc); and    -   X is selected from the group consisting of Cl, Br, I, and —OTs.

In another aspect, provided herein is a method of preparing aω-hydroxylated polyunsaturated fatty acid having a structure of:

or a salt thereof;the method includes:

-   -   i) forming a coupling reaction mixture comprising a copper ion,        an iodide salt, an alkali carbonate, an aprotic solvent, a        compound of Formula III-2a:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form the compound of Formula II-3a:

-   -   ii) forming a reaction mixture comprising a compound of Formula        II-3a, Lindlar catalyst, a N-containing 5- to 10-membered        heteroaryl compound, a C₂-C₆ alkene, and hydrogen, under        conditions suitable for hydrogenation to form a compound of        Formula I-3a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-3a and an alkali hydroxide under        conditions suitable for saponification to form 22-HdoHE;        wherein:    -   the copper ion is a copper (I) ion salt selected from the group        consisting of CuI, CuBr, CuCl, and Cu(OAc); and    -   X is selected from the group consisting of Cl, Br, I, and —OTs.

The iodide salt, the alkali carbonate, the aprotic solvent, theN-containing 5- to 10-membered heteroaryl compound, the C₂-C₆ alkene,and the alkali hydroxide are as defined and described herein.

In one selected embodiment, the copper (I) ion salt is CuI.

In one selected embodiments, the iodide salt is sodium iodide.

In one selected embodiment, the alkali carbonate is cesium carbonate.

In one selected embodiment, the aprotic solvent is dimethylformamide.

In one selected embodiment, the N-containing 5- to 10-memberedheteroaryl compound is pyridine.

In one selected embodiment, the C₂-C₆ alkene is 2-methyl-2-butene.

In one selected embodiment, the alkali hydroxide is NaOH. In anotherselected embodiment, the alkali hydroxide is LiOH.

In some selected embodiments, the hydrolysis reaction mixture furtherincludes methanol. In other selected embodiments, the hydrolysisreaction mixture further includes a mixture of water and THF.

In one selected embodiment, X is Br.

In one selected embodiment, the method including:

-   -   i) forming a coupling reaction mixture comprising CuI, NaI,        CsCO₃, DMF, a compound of Formula III-1a-Br:

-   -   -   a compound of Formula Iv-1 or Iv-2:

-   -   -   under conditions suitable to form a corresponding compound            of Formula II-1a or II-2a:

-   -   ii) forming a hydrogenation reaction mixture comprising the        compound of Formula II-1a or II-2a, Lindlar catalyst, pyridine,        2-methyl-2-butene, a hydrogenation solvent, and hydrogen, under        conditions suitable for hydrogenation to form a corresponding        compound of Formula I-1a or I-2a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-1a or I-2a, an alkali hydroxide, and a        hydrolysis solvent under conditions suitable for saponification        to form 20-HETE or 20-HEPE, or a salt thereof        Wherein:    -   the hydrogenation solvent is methanol, ethanol or ethyl acetate;    -   the alkali hydroxide is NaOH or LiOH; and the hydrolysis solvent        is methanol or a mixture of water and THF.

In another selected embodiment, the method including:

-   -   i) forming a coupling reaction mixture comprising CuI, NaI,        CsCO₃, DMF, a compound of Formula III-2a-Br:

and

-   -   a compound of Formula IV-2:

-   -   under conditions suitable to form the compound of Formula II-3a:

-   -   ii) forming a reaction mixture comprising a compound of Formula        II-3a, Lindlar catalyst, pyridine, 2-methyl-2-butene, a        hydrogenation solvent; and hydrogen, under conditions suitable        for hydrogenation to form a compound of Formula I-3a:

-   -   iii) forming a hydrolysis reaction mixture comprising the        compound of Formula I-3a, an alkali hydroxide, and a hydrolysis        solvent under conditions suitable for saponification to form        22-HdoHE or a salt thereof;        Wherein:    -   the hydrogenation solvent is methanol, ethanol or ethyl acetate;    -   the alkali hydroxide is NaOH or LiOH; and the hydrolysis solvent        is methanol or a mixture of water and THF.

In one selected embodiment, the hydrogenation solvent is methanol. Inone selected embodiment, the hydrogenation solvent is ethanol. Inanother selected embodiment, the hydrogenation solvent is ethyl acetate.

In one selected embodiment, the alkali hydroxide is NaOH. In anotherselected embodiment, the alkali hydroxide is LiOH.

In one selected embodiment, hydrolysis solvent is methanol. In anotherselected embodiment, hydrolysis solvent is a mixture of water and THF.

For the coupling reaction, other parameters (e.g., the molar equivalentsof the copper ion, the iodide salt, the alkali carbonate; theconcentration of the reaction; and reaction temperature) are asdescribed in detail above.

For the hydrogenation reaction, other parameters (e.g., reactiontemperature and hydrogen pressure) are as described in detail above.

For the hydrolysis reaction, the reaction temperature is as describedabove.

IV. Compounds

In one aspect, provided herein is a compound of Formula II:

wherein:

-   -   R is C₁-C₄ alkyl;    -   subscript m is 2 or 3;    -   subscript n is 0 or 1; and    -   bond a is a single bond or a triple bond.

In various embodiments, the compound of Formula II is a compound ofFormula II-1:

-   -   wherein R is as defined herein.

In various embodiments, the compound of Formula II is a compound ofFormula II-2:

-   -   wherein R is as defined herein.

In various embodiments, the compound of Formula II is a compound ofFormula II-3:

-   -   wherein R is as defined herein.

In some embodiments, R is methyl. In some embodiments, R is ethyl. Inother embodiments, R is isopropyl. In other embodiments, R is t-butyl.

In one selected embodiment, the compound of Formula II is a compound ofFormula II-1a, II-2a, or II-3a:

V. Pharmaceutical Compositions

In one aspect, provided herein is a pharmaceutical composition includinga ω-hydroxylated polyunsaturated fatty acid, or an ester form thereof,and a pharmaceutically acceptable excipient.

In some embodiments of the pharmaceutical compositions, theω-hydroxylated polyunsaturated fatty acid, or an ester form thereof, isincluded in a therapeutically effective amount.

In some embodiments, the ω-hydroxylated polyunsaturated fatty acid isselected from the group consisting of 20-HETE, 20-HEPE, and 22-HDoHE,and mixtures thereof. In some embodiments, the ω-hydroxylatedpolyunsaturated fatty acid is 20-HETE. In some embodiments, theω-hydroxylated polyunsaturated fatty acid is 20-HEPE. In otherembodiments, the ω-hydroxylated polyunsaturated fatty acid is 22-HDoHE.

In some other embodiments, the ester form of the ω-hydroxylatedpolyunsaturated fatty acid is a compound of formula I. In someembodiments, the ester form of the ω-hydroxylated polyunsaturated fattyacid is methyl ester. In some embodiments, the methyl ester form of theω-hydroxylated polyunsaturated fatty acid is a compound selected fromthe group consisting of Formula I-1a, I-2a, and I-3a. In someembodiments, the methyl ester form of the ω-hydroxylated polyunsaturatedfatty acid is a compound of Formula I-1a. In some embodiments, themethyl ester form of the ω-hydroxylated polyunsaturated fatty acid is acompound of Formula I-2a. In some embodiments, the methyl ester form ofthe ω-hydroxylated polyunsaturated fatty acid is a compound of FormulaI-3a.

In some embodiments of the pharmaceutical compositions, thepharmaceutical composition includes a second agent (e.g. therapeuticagent). In some embodiments of the pharmaceutical compositions, thepharmaceutical composition includes a second agent (e.g. therapeuticagent) in a therapeutically effective amount. In some embodiments of thepharmaceutical compositions, the second agent is an agent for treatingcancer. In some embodiments, the second agent is an anti-cancer agent.In other embodiments, the second agent is a chemotherapeutic.

VI. Methods of Use

In one aspect, provided herein is a method of treating cancer or maculardegeneration, the method including administering to a subject in needthereof an effective amount of a ω-hydroxylated polyunsaturated fattyacid, or the ester form thereof, or a pharmaceutical compositionthereof. In some embodiments, the ω-hydroxylated polyunsaturated fattyacid, or an ester form thereof, is included in a therapeuticallyeffective amount.

In some aspects, the method of treating cancer includes administering toa subject in need thereof an effective amount of a ω-hydroxylatedpolyunsaturated fatty acid, or the ester form thereof, or apharmaceutical composition thereof. In some embodiments, the cancer iscolorectal cancer. In some embodiments, the cancer is liver cancer. Insome embodiments, the cancer is hepatocellular cancer. In otherembodiments, the cancer is breast cancer. In other embodiments, thecancer is renal adenocarcinoma.

In some embodiments, the cancer is estrogen receptor positive breastcancer. In other embodiments, the cancer is estrogen receptor (ER)negative breast cancer. In some embodiments, the cancer is tamoxifenresistant breast cancer. In some embodiments, the cancer is HER2negative breast cancer. In other embodiments, the cancer is HER2positive breast cancer. In some embodiments, the cancer is low grade(well differentiated) breast cancer. In other embodiments, the cancer isintermediate grade (moderately differentiated) breast cancer. In stillother embodiments, the cancer is high grade (poorly differentiated)breast cancer. In some embodiments, the cancer is stage 0 breast cancer.In some embodiments, the cancer is stage I breast cancer. In someembodiments, the cancer is stage II breast cancer. In other embodiments,the cancer is stage III breast cancer. In other embodiments, the canceris stage IV breast cancer. In still other embodiments, the cancer istriple negative breast cancer.

In other aspects, the method of treating macular degeneration includesadministering to a subject in need thereof an effective amount of aω-hydroxylated polyunsaturated fatty acid, or the ester form thereof, ora pharmaceutical composition thereof. In some embodiments, theω-hydroxylated polyunsaturated fatty acid, or an ester form thereof, isincluded in a therapeutically effective amount.

In some embodiments, the ω-hydroxylated polyunsaturated fatty acidinhibits tumor growth, angiogenesis, lymphangiogenesis, or combinationsthereof.

Angiogenesis is the physiological process through which new bloodvessels form from pre-existing vessels. Angiogenesis is a normal andvital process in growth and development, as well as in wound healing andin the formation of granulation tissue. However, it is also afundamental step in the transition of tumors from a benign state to amalignant one, leading to the use of angiogenesis inhibitors in thetreatment of cancer. Vascular endothelial growth factor (VEGF) has beendemonstrated to be a major contributor to angiogenesis, increasing thenumber of capillaries in a given network. In some embodiments, theω-hydroxylated polyunsaturated fatty acid inhibits VEGF-inducedangiogenesis. In some embodiments, the ω-hydroxylated polyunsaturatedfatty acid inhibits tumor growth induced by VEGF.

Angiogenesis is defined as a new blood vessel sprouting frompre-existing vessels. This can be accomplished through endothelialsprouting or non-sprouting (intussusceptive) microvascular growth (IMG)The sprouting angiogenesis in tumor growth was reported to have thefollowing stages: 1) The basement membrane is locally degraded on theside of the dilated peritumoral postcapillary venule situated closed tothe angiogenic stimulus; 2) Interendothelial contacts are weakened andendothelial cells migrate into the connective tissue; 3) A solid cord ofendothelial cells form; 4) Lumen formation occurs proximal to themigrating front, contiguous tubular sprouts anastomose to formfunctionally capillary loops, parallel with the synthesis of the newbasement membrane and the recruitment of pericytes. In some embodiments,the ω-hydroxylated polyunsaturated fatty acid inhibits VEGF-inducedendothelial cell migration. In particular, the ω-hydroxylatedpolyunsaturated fatty acid inhibits VEGF-induced endothelial cellmigration in human umbilical vein endothelial cells (HUVECs).

Angiogenesis has been well established to play an essential role incancer, through providing nutrients and oxygen to the tumors tissues topromote tumor progression. In some embodiments, the ω-hydroxylatedpolyunsaturated fatty acid inhibits tumor growth. In particular, theω-hydroxylated polyunsaturated fatty acid inhibits tumor growth inducedby VEGF.

Lymphangiogenesis is the formation of lymphatic vessels frompre-existing lymphatic vessels in a method believed to be similar toangiogenesis (blood vessel development). Lymphangiogenesis has beenshown to play a critical role in tumor metastasis and many other humandiseases. Tumor-induced lymphangiogenesis is mediated by lymphangiogenicgrowth factors that are produced and secreted by the tumors themselves,stromal cells, tumor-infiltrating macrophages, or activated platelets.In recent years, experimentation has focused on the role of VEGF-C andVEGF-D in cancer progression. The overexpression of either vascularendothelial growth factor-C (VEGF-C) or VEGF-D in tumors significantlyincreased tumor-associated lymphatic vessel growth (primarily at thetumor margin) and increased incidence of lymph node metastasis. In someembodiments, the ω-hydroxylated polyunsaturated fatty acid inhibitslymphangiogenesis. In particular, the ω-hydroxylated polyunsaturatedfatty acid inhibits the vascular endothelial growth factor-C(VEGF-C)-induced tube formation in human dermal lymphatic endothelialcells (HMVEC-dLy).

In some embodiments, the ω-hydroxylated polyunsaturated fatty acid isselected from the group consisting of 20-HETE, 20-HEPE, and 22-HDoHE,and mixtures thereof. In some embodiments, the ω-hydroxylatedpolyunsaturated fatty acid is 20-HETE. In some embodiments, theω-hydroxylated polyunsaturated fatty acid is 20-HEPE. In otherembodiments, the ω-hydroxylated polyunsaturated fatty acid is 22-HDoHE.

In some other embodiments, the ester form of the ω-hydroxylatedpolyunsaturated fatty acid is a compound of formula I. In someembodiments, the ester form of the ω-hydroxylated polyunsaturated fattyacid is methyl ester. In some embodiments, the methyl ester form of theω-hydroxylated polyunsaturated fatty acid is a compound selected fromthe group consisting of Formula I-1a, I-2a, and I-3a. In someembodiments, the methyl ester form of the ω-hydroxylated polyunsaturatedfatty acid is a compound of Formula I-1a. In some embodiments, themethyl ester form of the ω-hydroxylated polyunsaturated fatty acid is acompound of Formula I-2a. In some embodiments, the methyl ester form ofthe ω-hydroxylated polyunsaturated fatty acid is a compound of FormulaI-3a.

In some embodiments, the method includes administering a second agent(e.g. therapeutic agent). In some embodiments, the method includesadministering a second agent (e.g. therapeutic agent) in atherapeutically effective amount. In some embodiments, the second agentis an agent for treating cancer. In other embodiments, the second agentis an anti-cancer agent. In other embodiments, the second agent is achemotherapeutic.

VII. Examples Example 1: General Chemical Methods Example 1.1: Chemicalsand Reagents

20-HETE used as a standard was purchased from Cayman Chemical (AnnArbor, Mich.). All other reagents and solvents were obtained fromcommercial suppliers and were used without further purification. Allreactions, unless otherwise described, were performed under an inertatmosphere of dry nitrogen.

Example 1.2: Instrumentation and Sample Analysis

Melting points were determined on an OptiMelt melting point apparatusand were uncorrected. ¹H NMR and ¹³C NMR spectra were recorded at 300and 75 MHz, respectively. Elemental analyses were determined at MidwestMicrolab, Indianapolis, Ind. Mass. spectra were measured by LC-MSequipped with a Waters 2790 and a Waters PDA 996 using electrospray (+)ionization. Flash chromatography was performed on silica gel.

Example 1.3: Synthesis Schemes for Preparing ω-HydroxylatedPolyunsaturated Fatty Acids

The syntheses of preparing ω-hydroxylated polyunsaturated fatty acids(i.e., 20-HETE, 20HEPE, and 22-HdoHE) are illustrated in Schemes 1 to 3.

Example 1.4: Synthesis Schemes for Preparing Intermediates

The compound of Formula III-1 can be prepared by a series of a copper(I)-mediated coupling reaction as described above and a conversion ofthe terminal —OH to Br (or Cl, I, —OTs, and —OTf). Accordingly, thepreparation of the compound of Formula III-1a-Br is described in Scheme4; and the preparation of the compound of Formula III-2a-Br is describedin Scheme 5.

The compound of Formula IV-1 or IV-2 can be prepared by a series of acopper (I)-mediated coupling reaction as described above and the removalof the silyl group. Accordingly, the preparation of the compound ofFormula IV-1 is described in Scheme 6; and the preparation of thecompound of Formula IV-2 is described in Scheme 7.

Example 2: Preparation of(5Z,8Z,11Z,14Z)-20-Hydroxyicosa-5,8,11,14-tetraenoic Acid (20-HETE)Example 2.1: Synthesis of Methyl hex-5-ynoate (1)

To a solution of 5-hexynoic acid (5 g, 44.6 mmol) in MeOH (400 mL) wasadded 10 drops of concentrated H₂SO₄ at room temperature. The reactionmixture was stirred for 2 days. The reaction mixture was quenched byadding 1 g of K₂CO₃ at room temperature. After 30 minutes, the solutionwas filtered and the filtrate was concentrated in vacuo. The remainedresidue was chromatographed on silica-gel to give the titled compound(3.94 g, 70%) as a colorless liquid. ¹H NMR (400 MHz, CDCl₃): δ 3.67 (s,3H), 2.45 (t, J=7 Hz, 2H), 2.26 (dt, J=7 and 3 Hz, 2H), 1.96 (t, J=3 Hz,1H), 1.84 (p, J=7 Hz, 2H).

Example 2.2: Synthesis of Methyl 10-hydroxydeca-5,8-diynoate (2)

To a solution of methyl hex-5-ynoate (1) (4.4 g, 35 mmol) in DMF (50 mL)were added CuI (6.6 g, 35 mmol), NaI (5.2 g, 35 mmol), Cs₂CO₃ (11.4 g,35 mmol), and 4-chlorobutyn-1-ol (3 mL, 35 mmol) at room temperature.The reaction mixture was stirred overnight. The reaction was quenched byadding saturated NH₄Cl and extracted with Et₂O (3×50 mL). The combinedorganic layers were washed with saturated NH₄C₁, aq. Na₂S₂O₃, and water,successively. After filtering, the solvents were removed in vacuo andthe residue was purified by column chromatography to give the titledcompound (4.2 g, 82%) as a clear oil. ¹H NMR (400 MHz, CDCl₃): δ 4.23(t, J=2 Hz, 2H), 3.66 (s, 3H), 3.16 (p, J=2 Hz, 2H), 2.42 (t, J=7 Hz,2H), 2.22 (tt, J=7 and 2 Hz, 2H), 1.83 (s, 1H), 1.80 (p, J=7 Hz, 2H).

Example 2.3: Synthesis of Methyl 10-bromodeca-5,8-diynoate (3)

To a solution of methyl 10-hydroxydeca-5,8-diynoate (2) (2.55 g, 13.1mmol) and tetrabromomethane (5.6 g, 16.9 mmol) in 130 mL ofdichloromethane was added triphenylphosphine (4.6 g, 17.5 mmol) at 0° C.After stirring for 2 hours, the solvent was removed in vacuo. To theresidue was added hexanes and the precipitates were removed by filter.The filtrate was concentrated in vacuo and purified by columnchromatography to give the titled compound as a colorless oil (2.1 g,81% yield). ¹H NMR (400 MHz, CDCl₃): δ 3.90 (t, J=2 Hz, 2H), 3.67 (s,3H), 3.20 (p, J=2 Hz, 2H), 2.42 (t, J=7 Hz, 2H), 2.23 (tt, J=7 and 2 Hz,2H), 1.81 (p, J=7 Hz, 2H).

Example 2.4: Synthesis of 10-(Trimethylsilyl)deca-6,9-diyn-1-ol (5)

To a solution of 6-heptyn-1-ol (1.2 g, 10.5 mmol) in DMF (15 mL) wereadded CuI (2.0 g, 10.5 mmol), NaI (1.6 g, 10.5 mmol), Cs₂CO₃ (3.4 g,10.5 mmol), and ethynyltrimethylsilane (2 g, 10.5 mmol) at roomtemperature. The reaction mixture was stirred overnight. The reactionwas quenched by adding saturated NH₄Cl and extracted with Et₂O (3×50mL). The combined organic layers were washed with saturated NH₄Cl, aq.Na₂S₂O₃, and water, successively. After filtering, the solvents wereremoved in vacuo and the residue was purified by column chromatographyto give the titled compound (1.6 g, 69%) as a colorless oil. ¹H NMR (400MHz, CDCl₃): δ 3.63 (t, J=7 Hz, 2H), 3.17 (t, J=2 Hz, 2H), 2.17-2.10 (m,2H), 1.71 (s, 1H), 1.58-1.37 (m, 6H), 0.14 (s, 9H).

Example 2.5: The Synthesis of Deca-6,9-diyn-1-ol (6)

To a solution of 10-(trimethylsilyl)deca-6,9-diyn-1-ol (5) (1.1 g, 4.8mmol) in 40 mL of THF were added tetrabutylammoniumfloride (1M in THF,4.8 mL) and acetic acid (0.16 mL) simultaneously dropwise at roomtemperature. After stirring 1 h, the reaction mixture was extracted withEt₂O (2 times). The combined organic layers were washed with water andbrine. After drying with MgSO₄, the solvents were removed in vacuo. Theremained residue was purified by column chromatography to give thetitled compound (0.13 g, 81% yield) as a clear oil. ¹H NMR (400 MHz,CDCl₃): δ 3.64 (t, J=7 Hz, 2H), 3.15-3.13 (m, 2H), 2.20-2.15 (m, 2H),2.39 (s, 1H), 2.06 (t, J=3 Hz, 1H), 1.61-1.39 (m, 6H).

Example 2.6: Synthesis of Methyl 20-hydroxyicosa-5,8,11,14-tetraynoate(17)

To a solution of deca-6,9-diyn-1-ol (6) (0.5 g, 3.3 mmol) in DMF (10 mL)were CuI (0.63 g, 3.3 mmol), NaI (0.49 g, 3.3 mmol), Cs₂CO₃ (1.1 g, 3.3mmol), and methyl 10-bromodeca-5,8-diynoate (3) (0.93 g, 3.6 mmol) atroom temperature. The reaction mixture was stirred overnight. Thereaction was quenched by adding saturated NH₄Cl and extracted with Et₂O(3×20 mL). The combined organic layers were washed with saturated NH₄C₁,aq. Na₂S₂O₃, and water, successively. After filtering, the solvents wereremoved in vacuo and the residue was purified by column chromatographyto give the titled compound (0.43 g, 40%) as a clear oil. ¹H NMR (400MHz, CDCl₃): δ 3.68 (s, 3H), 3.65 (t, J=7 Hz, 2H), 3.17-3.11 (m, 6H),2.43 (t, J=7 Hz, 2H), 2.26-2.15 (m, 4H), 1.84-1.76 (m, 2H), 1.61-1.41(m, 6H), 1.24 (s, 1H). MS (ESI) m/z: 335.59 (M+H⁺).

Example 2.7: Synthesis of Methyl(5Z,8Z,11Z,14Z)-20-hydroxyicosa-5,8,11,14-tetraenoate (18)

Partial Hydrogenation Reaction of Compound 17 to Compound 18:

It was found that the partial hydrogenation reaction of compound 17 wasdifficult to control without further reducing to alkane underconventional partial hydrogenation conditions (i.e., Lindlar catalystthat is further poisoned with addition of quinoline, pyridine, orethylenediamine). To overcome the technical difficulties, it was testedwhether an extra additive, 2-methyl-2-buene, can prevent undesiredadditional hydrogenation of compound 17. Therefore compound 17 wassubject to partially hydrogenate with Lindlar catalyst, pyridine in EtOHin the presence of excess 2-methyl-2-butene. The desired compound 18,20-HETE methyl ester, was obtained exclusively. In the presence ofexcess amounts of 2-methyl-2-butene, no further hydrogenation ofcompound 18 was occurred.

To confirm whether the method can be repeatable and scalable, thereaction was performed in duplicate (entry I, Table 1) and in a 5-foldscale (entry II, Table 1). With the exception that the reaction time wasprolonged with the reaction scale, the reaction was repeatable. Withthese results in hand, the partial hydrogenation of both compounds 19(see Example 3.6) and 21 (see Example 4.7) was successful in reducingmultiple triple bonds to the desired cis-double bonds of 20, and 22,respectively.

TABLE 1 Partial hydrogenation of 17 to 18 (see Scheme 1) Scale Compound17 Compound 18 Time (h)^(a) Yield (%) I 100 mg 40 mg 18  43^(b) (0.31mmol) II 500 mg 250 mg 28 48 (1.55 mmol) ^(a)Reaction conditions:Lindlar catalyst (1 eq), 2-methyl-2-butene:EtOH:pyridine (4:4:1), roomtemperature, H₂ gas (1 atm). ^(b)Average yield in duplicates.

To a solution of methyl 20-hydroxyicosa-5,8,11,14-tetraynoate (17) (0.1g, 0.31 mmol) in 4:1 mixture of EtOH:pyridine and 2 mL of2-methyl-2-butene was added Lindlar catalyst (0.1 g) at roomtemperature. The reaction mixture was stirred overnight under hydrogenatmosphere. The reaction mixture was filtered and washed with EtOAc. Thefiltrate was washed with water. The organic layer was dried with MgSO₄and filtered. After removing the solvents in vacuo, the residue waspurified by column chromatography to give the titled compound ascolorless oil (48 mg, 46% yield). ¹H NMR (400 MHz, CDCl₃): δ 5.42-5.30(m, 8H), 3.66 (s, 3H), 3.63 (t, J=7 Hz, 2H), 2.86-2.76 (m, 6H), 2.31 (t,J=8 Hz, 2H), 2.13-2.02 (m, 5H), 1.70 (p, J=8 Hz, 2H), 1.61-1.51 (m, 2H),1.42-1.32 (m, 4H). MS (ESI) m/z: 321.55 (M+H⁺).

Example 2.8: Synthesis of(5Z,8Z,11Z,14Z)-20-Hydroxyicosa-5,8,11,14-tetraenoic acid (20-HETE)

To a solution of methyl(5Z,8Z,11Z,14Z)-20-hydroxyicosa-5,8,11,14-tetraenoate (18) (16 mg, 0.05mmol) in 2 mL of THF was added 0.1 M LiOH in water (0.5 mL) at roomtemperature. The reaction mixture was stirred overnight and extractedwith EtOAc (2×). The combined organic layers were dried with MgSO₄ andfiltered. The filtrate was concentrated in vacuo and purified by columnchromatography to give the titled compound as colorless oil (34 mg, 88%yield). ¹H NMR (400 MHz, CDCl₃): δ 5.45-5.30 (m, 8H), 3.66 (t, J=7 Hz,2H), 2.87-2.77 (m, 6H), 2.35 (t, J=7 Hz, 2H), 2.17-2.02 (m, 5H), 1.71(p, J=7 Hz, 2H), 1.61-1.52 (m, 2H), 1.42-1.33 (m, 4H). MS (ESI) m/z:319.2 (M⁻).

Example 3: Preparation of(5Z,8Z,11Z,14Z,17Z)-20-Hydroxyicosa-5,8,11,14,17-pentaenoic Acid(20-HEPE) Example 3.1: Synthesis of 6-(Trimethylsilyl)hexa-2,5-diyn-1-ol(8)

The titled compound (2.6 g, 77% yield as a colorless oil) wassynthesized in a manner similar to the synthesis of the compound (5)using 4-chloro-2-butyn-1-ol (2.1 g, 20 mmol). ¹H NMR (300 MHz, CDCl₃): δ4.26 (t, J=2 Hz, 2H), 3.25 (t, J=2 Hz, 2H), 1.65 (s, 1H), 0.16 (s, 9H).

Example 3.2: Synthesis of 6-(trimethylsilyl)hexa-2,5-diyn-1-yl4-methylbenzene-1-sulfonate (9)

To a solution of 6-(trimethylsilyl)hexa-2,5-diyn-1-ol (8) (2 g, 12 mmol)and p-toluenesulfonyl chloride (2.75 g, 14.4 mmol) in dichloromethanewere added Et₃N (2.5 mL, 18 mmol) and a catalytic amounts of DMAP at 0°C. The reaction mixture was warmed up to room temperature and stirredovernight. The reaction mixture was washed with water and the organiclayer was dried with MgSO₄. After filtering, the solvent was removed invacuo. The residue was purified by column chromatography to give thetitled compound (2.0 g, 51%) as white solid. ¹H NMR (300 MHz, CDCl₃): δ7.82 (d, J=8 Hz, 2H), 7.35 (d, J=8 Hz, 2H), 4.70 (t, J=2 Hz, 2H), 3.11(t, J=2 Hz, 2H), 2.46 (s, 3H), 0.16 (s, 9H).

Example 3.3: Synthesis of 10-(Trimethylsilyl)deca-3,6,9-triyn-1-ol (10)

The titled compound (0.63 g, 50% yield as a colorless oil) wassynthesized in a manner similar to the synthesis of the compound (5)using 3-butyn-1-ol (0.41 g, 5.8 mmol) and the compound (9) (1.9 g, 5.8mmol). ¹H NMR (400 MHz, CDCl₃): δ 3.70 (t, J=6 Hz, 2H), 3.22-3.19 (m,2H), 3.18-3.14 (m, 2H), 2.47-2.41 (m, 2H), 1.77 (s, 1H), 0.16 (s, 9H).

Example 3.4: Synthesis of deca-3,6,9-triyn-1-ol (11)

The titled compound (0.22 g, 85% as clear oil) was prepared in a mannersimilar to the synthesis of the compound (6) using the compound (10)(0.4 g, 1.8 mmol). ¹H NMR (400 MHz, CDCl₃): δ 3.71 (t, J=6 Hz, 2H),3.19-3.13 (m, 4H), 2.47-2.42 (m, 2H), 2.09 (s, 1H), 2.07 (t, J=3 Hz,1H).

Example 3.5: Synthesis of Methyl20-hydroxyicosa-5,8,11,14,17-pentaynoate (19)

The titled compound (0.15 g, 40% as yellowish oil) was prepared in asimilar to the synthesis of the compound (17) from the compound (11)(0.17 g, 1.2 mmol) and the compound (3) (0.33 g, 1.3 mmol). ¹H NMR (400MHz, CDCl₃): δ 3.70 (t, J=6 Hz, 2H), 3.67 (s, 3H), 3.17-3.10 (m, 8H),2.48-2.36 (m, 4H), 2.26-2.19 (m, 2H), 1.81 (p, J=7 Hz, 2H).

Example 3.6: Synthesis of Methyl(5Z,8Z,11Z,14Z,17Z)-20-hydroxyicosa-5,8,11,14,17-pentaenoate (20)

The titled compound (46 mg, 45% as yellowish oil) was prepared in asimilar to the synthesis of the compound (18) from the compound (19)(0.1 g, 0.31 mmol) except the reaction mixture was stirred for 3 days inEtOAc instead of EtOH. ¹H NMR (400 MHz, CDCl₃): δ 5.62-5.30 (m, 10H),3.70-3.60 (m, 2H), 3.67 (s, 3H), 2.90-2.75 (m, 8H), 2.40-2.29 (m, 4H),2.11 (dd, J=14 and 7 Hz, 2H), 1.71 (p, J=7 Hz, 2H), 1.58 (s, 1H). MS(ESI) m/z: 381.60 (M+Na⁺).

Example 3.7: The synthesis of(5Z,8Z,11Z,14Z,17Z)-20-Hydroxyicosa-5,8,11,14,17-pentaenoic acid(20-HEPE)

The titled compound (8 mg, 50% as yellowish oil) was prepared in asimilar to the synthesis of the compound 20-HETE from the compound (20)(16.7 mg, 0.05 mmol). ¹H NMR (400 MHz, CDCl₃): δ 5.63-5.31 (m, 10H),3.77-3.61 (m, 2H), 2.94-2.75 (m, 8H), 2.43-2.32 (m, 4H), 2.14 (dd, J=14and 7 Hz, 2H), 1.71 (q, J=7 Hz, 2H). MS (ESI) m/z: 317.2 (M).

Example 4: preparation of(4Z,7Z,10Z,13Z,16Z,19Z)-22-Hydroxydocosa-4,7,10,13,16,19-hexaenoic acid(22-HDoHE) Example 4.1: Synthesis of Methyl pent-4-ynoate (12)

The titled compound (2.86 g, 50% as yellowish oil) was prepared in asimilar to the synthesis of the compound (18) from the compound (19) (5g, 51 mmol). ¹H NMR (400 MHz, DMSO-d₆): δ 3.62 (s, 3H), 2.81-2.80 (m,1H), 2.54-2.49 (m, 2H), 2.42-2.39 (m, 2H).

Example 4.2: Synthesis of Methyl 9-hydroxynona-4,7-diynoate (13)

The titled compound (1.35 g, 75% as yellowish oil) was prepared in asimilar to the synthesis of the compound (2) from the compound (12)(1.12 g, 10 mmol). ¹H NMR (400 MHz, CDCl₃): δ 4.26 (t, J=2 Hz, 2H), 3.70(s, 3H), 3.17 (p, J=2 Hz, 2H), 2.55-2.45 (m, 4H). OH peak is missing.

Example 4.3: Synthesis of Methyl 9-bromonona-4,7-diynoate (14)

The titled compound (0.65 g, 75% yield) was prepared in a similar to thesynthesis of the compound (13) (0.65 g, 3.6 mmol). ¹H NMR (400 MHz,CDCl₃): δ 3.90 (t, J=2 Hz, 2H), 3.69 (s, 3H), 3.19 (p, J=2 Hz, 2H),2.55-2.45 (m, 4H).

Example 4.4: The synthesis of Methyl 12-hydroxydodeca-4,7,10-triynoate(15)

The titled compound (0.55 g, 94% as yellowish oil) was prepared in asimilar to the synthesis of the compound (2) from the compound (14)(0.65 g, 2.67 mmol). ¹H NMR (400 MHz, CDCl₃): δ 4.26-4.24 (m, 2H), 3.69(s, 3H), 3.21-3.17 (m, 2H), 3.13-3.09 (m, 2H), 2.55-2.44 (m, 4H), 1.71(s, 1H).

Example 4.5: Synthesis of Methyl 12-bromododeca-4,7,10-triynoate (16)

The titled compound (0.35 g, 50% yield) was prepared in a similar to thesynthesis of the compound (15) (0.54 g, 2.5 mmol). ¹H NMR (300 MHz,CDCl₃): δ 3.90 (t, J=2 Hz, 2H), 3.69 (s, 3H), 3.22 (q, J=2 Hz, 2H), 3.12(t, J=2 Hz, 2H), 2.56-2.44 (m, 4H).

Example 4.6: Synthesis of Methyl22-hydroxydocosa-4,7,10,13,16,19-hexaynoate (21)

The titled compound (0.1 g, 58% as yellowish oil) was prepared in asimilar to the synthesis of the compound (17) from the compound (11)(0.073 g, 0.5 mmol) and the compound (16) (0.15 g, 0.55 mmol). ¹H NMR(400 MHz, CDCl₃): δ 3.71 (t, J=6 Hz, 2H), 3.70 (s, 3H), 3.17-3.10 (m,10H), 2.56-2.42 (m, 6H), 1.74 (s, 1H).

Example 4.7: The synthesis of Methyl(4Z,7Z,10Z,13Z,16Z,19Z)-22-hydroxydocosa-4,7,10,13,16,19-hexaenoate (22)

The titled compound (0.04 mg, 50% as yellowish oil) was prepared in asimilar to the synthesis of the compound (18) from the compound (21)(0.08 g, 0.23 mmol) except the reaction mixture was stirred for 3 daysin EtOAc instead of EtOH. ¹H NMR (400 MHz, CDCl₃): δ 5.60-5.29 (m, 1H),3.67 (s, 1H), 3.66 (t, J=7 Hz, 2H), 2.90-2.78 (m, 1H), 2.42-2.32 (m,1H), 1.51 (s, 1H). MS (ESI) m/z: 381.60 (M+H⁺).

Example 4.8: Synthesis of(4Z,7Z,10Z,13Z,16Z,19Z)-22-Hydroxydocosa-4,7,10,13,16,19-hexaenoic acid(22-HDoHE)

The titled compound (7.5 mg, 78.5% as yellowish oil) was prepared in asimilar to the synthesis of the compound 20-HETE from the compound (20)(10 mg, 0.028 mmol). ¹H NMR (400 MHz, CDCl₃): δ 5.60-5.29 (m, 1H), 3.67(s, 1H), 3.66 (t, J=7 Hz, 2H), 2.90-2.78 (m, 1H), 2.42-2.32 (m, 1H),1.51 (s, 1H). MS (ESI) m/z: 343.2 (M).

Example 5: Characterization of Prepared 20-HETE, 20-HEPE, and 22-HDoHE

The structure and purity of purified 20-HETE were further assessed byLC-MS/MS. The hydrolyzed sophorolipids or isolated 20-HETE was dissolvedin methanol or acetonitrile to prepare a 1 mg/ml solution, then diluted1,000-4,000 times for LC-MS/MS analysis. The solutions were injectedinto a LC-MS/MS system, including Agilent 1200SL (Santa Clara, Calif.)system coupled to AB Sciex 4000 QTrap system (Foster City, Calif.). TheLC/MS/MS method was described in Reference 37. The mass spectrometer wasoperated under negative electrospray mode. The MRM transition for20-HETE, 20-HEPE, and 22-HDoHE were 319.2/275.2, 317.2/255.0, and343.2/281.1, respectively. Other parameters for 20-HETE were DP-60 V,CE-26, and CXP-8. In addition, the identification of synthesized 20-HETEwas implemented by comparing with 20-HETE and 20-HETE-d₆ standard.

The purity and identification of 20-HETE, 20-HEPE, and 22-HDoHE,evaluated by LC-MS/MS, is shown in FIG. 2. ¹H NMR spectra of 20-HETE,20-HEPE, and 22-HDoHE are shown in FIG. 3. As shown in FIG. 2A, thesynthesized 20-HETE have the same retention time and the mass comparedto the commercial 20-HETE (>98% of purity). The purity of 20-HETE (95.1%of purity), 20-HEPE (91.3% of purity) or 22-HDoHE (94.6% of purity) wasdetermined by comparison of observed mass and calculated mass. 20-HETEwas found to have a purity of 95.1%; 20-HEPE was found to have a purityof 91.3%; and 22-HDoHE was found to have a purity of 94.6%; accordingly.

Example 6: Study of 20-HETE, 20-HEPE, and 22-HDoHE for their Activationof TRPV1 In Vitro

mTRPV1 Plasmid and HEK-293 Cells:

The mTRPV1 plasmid that contains a GFP tag was a generous gift fromProfessor Jie Zheng at University of California, Davis. The HEK-293cells were cultured in a poly-D-lysine coated T75 dish at an initialdensity of 4×10⁵ cells/dish and maintained in culture medium (DMEM+10%FBS+100 units/ml penicillin, 0.1 mg/ml streptomycine) in an incubator at37° C. with 5% CO₂ and 95% humidity. At 50% confluence, the HEK-293cells were transfected with 10 μg of mTRPV1 plasmid using Lipofectamine2000 (Invitrogen Corporation, Carlsbad, Calif., USA) in antibiotic—aswell as serum-free culture medium. After 6 hour incubation, theserum-free medium was replaced with normal culture medium and the cellswere cultured for another 48 hours. The transfection efficiency wasdetermined by examined the GFP expression as well as the Ca²⁺ responseafter exposure to capsaicin.

The HEK-293 cells transiently transfected mTRPV1 were planted ontopoly-D-lysine (Sigma-Aldrich, St. Louis, Mo., USA) coated black-well,clear-bottom, 96-well plates (Corning, Coring, N.Y., USA) at an initialdensity of 40,000/well. The cells were then cultured in DMEM medium for18-24 hours as described previously (Ref. 38). Briefly, the growthmedium was removed and replaced with dye loading buffer (100 μl/well)containing 4 μM fluo-4 and 0.5 mg/ml BSA in Locke's buffer (in mM: 8.6HEPES, 5.6 KCl, 154 NaCl, 5.6 Glucose, 1.0 MgCl₂, 2.3 CaCl₂), 0.0001glycine, pH 7.4). After 1 hour incubation in dye loading buffer, cellswere washed four times in fresh Locke's buffer and transferred to theplate chamber of FLIPR^(TETRA) (Molecular Devices, Sunnyvale, Calif.,USA). The final volume of Locke's buffer in each well was 150 μl. Thecells were excited at 488 nm and Ca²⁺ bound fluo-4 emission at 535 nmwas recorded. Fluorescence readings were taken every 1 second for 120 sto establish the baseline. Different concentrations (4×) of compoundswere added to the cells from a compound plate in a volume of 50 μl at arate of 30 μl/s using a programmable robotic system. The fluorescencesignals were recorded for an additional 380 seconds. All the experimentswere performed at room temperature. The Ca²⁺ data were represented asFluo-4 fluorescence units (F/F₀). For quantifying the Ca²⁺ response, thearea under the curve (AUC) was used. Graphing and statistical analyseswere performed using GraphPad Prism software (GraphPad Software Inc.,San Diego, Calif.). Statistical significance was determined by an ANOVAand, where appropriate, a Dunnett's multiple comparison test and pvalues below 0.05 was considered statistically significant.

In vitro Study of ω-Hydroxylated Polyunsaturated Fatty Acids forActivation of mTRPV1

The influence of 20-HETE, 20-HEPE, and 22-HDoHe on mTRPV1 was measuredin HEK293 cells heterologously expressed mTRPV1 using calcium influxassay. Capsaicin was applied to stimulate the Ca²⁺ influx in mTRPV1expressed HEK293 cells as a positive control, shown in FIG. 4. It wasfound that 22-HDoHE (˜3 μM concentration) produced significant Ca²⁺influx and 20-HEPE produced significant Ca²⁺ influx at 10 μM. However,20-HETE produced marginal Ca²⁺ response at the concentrations examined(see FIG. 4). It is worth noting that 20-HETE at 10 μM concentrationactivates hTRPV1 in a previous study (Ref 24). The discrepancy betweenprevious study and current study is likely due to the fact that themTRPV1 was used in the current study, while the hTRPV1 was used in theprevious study. Nevertheless, the current data has demonstrated thatboth 20-HEPE and 22-HDoHE may be more potent (or more efficacious) tostimulate mTRPV1 than 20-HETE does.

As shown in FIG. 4, 20-HETE does not appear to be an mTRPV1 activator ator below concentration of 10 uM. Similarly, 20-HEPE and 22-HDoHE did notactivate TRPV1 channels at low doses. However the ω-3 metabolitesdemonstrated activity at the highest dose of 10 μM but with far lesssignal than a low dose of the classical agonist capsaicin.

Example 7: Study of 20-HETE, 20-HEPE, and 22-HDoHE for Inhibition ofCOXs

In addition, 20-HETE, but not 20-HEPE or 22-HDoHE, has been known to bemetabolized by cyclooxygenases (COXs) to form 20-hydroxy PGE2 (20-OHPGE2), which is merely known as a vasodilator (see Ref 29)

Percent inhibition at 100 μM of 20-HETE, 20-HEPE, and 22-HDoHe againstovine COX-1 and human COX-2 was determined using a COX FluorescentInhibitor Screening Assay Kit (catalog number 700100, Cayman Chemical,Ann Arbor, Mich.) according to the manufacturer's instructions. Briefly,to a series of supplied reaction buffer solutions (150 μl, 100 mMTris-HCl, pH 8.0) with either COX-1 or COX-2 (10 μl) enzyme in thepresence of Heme (10 μl) and fluorometric substrate (10 μl) were added10 μl of 100 μM concentration of the test compound solutions. Thereactions were initiated by adding 10 μl of ARA solution and thenincubated for two minutes at room temperature. Percent inhibition wascalculated by comparison from the 100% initial activity sample value (noinhibitor). Results were determined by the test run in triplicate, shownin Table 2. No COX-2 activity changes have been found up to 100 μMconcentrations, as shown in FIG. 5.

TABLE 2 Inhibition of 20-HETE, 20-HEPE, and 22- HDoHe against COX-1 andCOX-2 enzymes % inhibition at 100 μM COX-1 COX-2 Indomethacin 87 —celecoxib — 99 20-HETE 27.5 0 20-HEPE 37.1 0 22-HDoHe 38.1 1

Example 8: Study of Mechanical Allodynia In Vivo Induced by 20-HETE,20-HEPE, and 22-HDoHE

Male Sprague-Dawley rats weighing approximately 250-300 grams wereobtained from Charles River Laboratories. On the day of the test, ratswere brought to the testing apparatus and allowed to acclimate. Theapparatus is a raised metal mesh platform with clear acrylic chambers toenclose rats but allow them to move freely. After 30 minutes the ratswere tested with a von Frey aesthesiometer for their non-treatedbaseline withdrawal thresholds using a rigid tip on the plantar surfaceof the rat hind paw though the mesh floor. The scores are grams of forceapplied to the hind paw required to elicit a withdrawal. Thepretreatment baseline score was considered the pain-free baseline (BL)and was assigned 100% for further response calculations.

For the pain assay, 20-HETE methyl ester, 20-HEPE methyl ester, or22-HDoHE methyl ester, formulated in 10% EtOH in saline, were injectedintraplantar in naïve rats. Withdrawal thresholds were measured at 15,30, 60, 90, 120 and 150 minutes post injection. Reported scores arecalculated as a percent of the pretreatment baseline score with anaverage of 6 rats (3 trials per rat) with SEM for the group.

The methyl esters compounds of 20-HETE, 20-HEPE and 22-HDoHE were testedfor their ability to induce mechanical allodynia in vivo. For the assay,naïve rats were injected intraplantar with 1 μg of each ester and testedwith an electronic von Frey aesthesiometer. The results are shown inFIG. 6A. 20-HETE decreased nociceptive thresholds acutely in naïve ratsand in a dose-response manner only with ipsilateral administration, asshown in FIG. 6B. However neither 20-HEPE nor 22-HDoHE induced a similardecrease in thresholds.

As shown in Example 6, both 20-HEPE and 22-HDoHE are potent TRPV1agonist but not a pain inducer like 20-HETE in vivo.

Example 9: The Effect of 22-HDoHE on Angiogenesis

Anti-Angiogenic Action of 22-HDoHE In Vivo:

Angiogenesis, the process of formation of new blood vessels frompre-existing vessels, has been established to be critical for multiplehuman diseases including cancer. The effect of 22-HDoHE on angiogenesiswas examined using a Matrigel plug assay in C57BL/6 mice, as shown inFIG. 7A. Vascular endothelial growth factor (VEGF) is the most importantangiogenesis inducer. Implantation of Matrigel plugs containing 100 ngVEGF in mice triggered a robust angiogenic response, while ω-addition of22-HDoHE in the Matrigel almost abolished VEGF-induced angiogenesis,demonstrating the anti-angiogenic action of 22-HDoHE in vivo, as shownin FIG. 7B.

Anti-Angiogenic Action of 22-HDoHE In Vitro:

It was tested whether 22-HDoHE has direct anti-angiogenic actions inprimary endothelial cells, using a VEGF-induced endothelial cellmigration in human umbilical vein endothelial cells (HUVECs). At a doserange of 1-3 μM, 22-HDoHE significantly inhibited VEGF-induced HUVECsmigration, demonstrating its anti-angiogenic activity in vitro. Theresults are shown in FIG. 8.

Example 10: The Effect of 22HDoHE on Primary Tumor Growth

Angiogenesis has been well established to play an essential role incancer, through providing nutrients and oxygen to the tumors tissues topromote tumor progression. The effect of 22HDoHE on primary tumor growthin mice was studied using a highly aggressive B16F10 melanoma model inC57BL/6 mice. Treatment with 0.5 mg/kg/day 22-HDoHE caused anapproximate 50% reduction of B16F10 tumor growth in mice, demonstratingits anti-cancer effects in vivo. The results are shown in FIG. 9.

Example 11: The Effect of 22HDoHE on Lymphangiogenesis

Lymphangiogenesis has been shown to play a critical role in tumormetastasis and many other human diseases. The effect of 22-HDoHE onlymphangiogenesis was evaluated using a vascular endothelial growthfactor-C (VEGF-C)-induced tube formation in human dermal lymphaticendothelial cells (HMVEC-dLy). The result showed that 22-HDoHEsignificantly inhibited VEGF-C-induced tube formation in HMVEC-dLy cellsin a dose-dependent manner, demonstrating its anti-lymphangiogenicactivity in vitro. The results are shown in FIG. 10.

The effect of 22-HDoHE on cellular migration of lymphatic endothelialcells was tested, and found it can significantly inhibit completemedium-induced HMVEC-dLy migration in a Boyden chamber assay (FIG. 11).

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Although the teachings have been described with respect to variousembodiments, it should be realized that these teachings are also capableof a wide variety of further and other embodiments within the spirit andscope of the appended claims.

What is claimed is:
 1. A method of treating cancer or maculardegeneration, the method comprising administering to a subject in needthereof an effective amount of a ω-hydroxylated polyunsaturated fattyacid, or an ester form thereof, or a pharmaceutical composition thereof,wherein the ω-hydroxylated polyunsaturated fatty acid is selected fromthe group consisting of 20-HEPE, 22-HdoHE, or mixtures thereof.
 2. Themethod of claim 1 wherein the ω-hydroxylated polyunsaturated fatty acidinhibits tumor growth, angiogenesis, lymphangiogenesis, or combinationsthereof.